Differentiating infected from vaccinated animals is known as the DIVA strategy. Vaccination is primarily performed using killed whole virus-adjuvanted vaccines. Proper vaccination may reduce or prevent clinical signs, reduce virus shedding in infected birds, and increase the resistance to infection.
One limitation of killed whole virus-adjuvanted vaccines is that vaccinated birds cannot be distinguished serologically from naturally infected birds using commonly available diagnostic tests. Thus detection of avian influenza becomes much more difficult and often results in trade restrictions because of the inability of the DIVA strategy to distinguish between vaccinated and infected individuals.
The most common method to distinguish vaccinated individuals from infected individuals is the use of unvaccinated sentinels. A second approach is the use of subunit vaccines targeted to the hemagglutinin protein allowing serologic surveillance to the internal proteins. A third strategy is to vaccinate with a homologous hemagglutinin to the circulating field strain, using a heterologous neuraminidase subtype. Serologic detection can then be performed for the homologous neuraminidase subtype as evidence of natural infection. A fourth strategy is to measure the serologic response to the nonstructural protein 1 (NS1). The NS1 protein is produced in large quantities in infected cells, but it is not packaged in the virion. Since killed virus-adjuvanted vaccines for influenza are primarily made with whole virions, a differential antibody response can be seen between naturally infected and vaccinated animals. However, poultry vaccines are not highly purified, and they contain small amounts of the NS1 protein.
Although vaccinated chickens will produce low levels of antibody to the NS1 protein, virus infected chickens will produce higher levels of NS1 antibody, and the two groups can be differentiated. All four DIVA strategies have advantages and disadvantages, and further testing is needed to identify the best strategy to make vaccination a more viable option for avian influenza.
In 2005, Terrence M. Tumpey produced a recombinant NS1 protein in Escherichia Coli and developed an ELISA and western blot method to measure the antibody in infected poultry against the NS1 protein. In this research, the reaction band in western blot was shown. Tumpey even introduced two sequences of antigenic peptides in the NS1 protein which have an important role in producing the antibody.
They developed an ELISA system with these peptides to differentiate between the infected and vaccinated poultry, but because of the small size of the peptides, it was not possible to develop a western blot method. ELISA is a colorimetric test and it must be confirmed with other tests like western blot.
In 2007, Laurence Duchesne developed a western blot method for detection of low molecular weight peptides. They made a coated metal like membrane to keep the peptides on the membrane so the peptides will not be washed out through the membrane during washing.
In this invention, the conserved peptide of the NS1 protein is applied to detect the antibody against peptide antigen in infected poultry to differentiate infected poultry from vaccinated ones. This peptide will be transferred from the gel to the silver nanoparticle-coated membrane. This will be done through a western blot process and the peptide antigen will react with the antibody which is present in infected serum. In fact, this methodology may be generalized to any protein having one or more conserved peptide regions and may thus be used in other contexts wherein it is necessary to differentiate between vaccinated, naturally resistant and infected individuals.